1. Field of the Invention
This present invention relates to the analysis and processing of biological cells and other particles and more particularly to a method and device for analyzing and processing biological cells using a laser diode with an external cavity.
2. Discussion of the Background
In recent years, rapid in-situ monitoring of biological cells and detection of ultra-small-volume samples, and even of single cells, are in great demand (1). Those analyses and processing may include the determination of morphologic characteristics of cells and biophysical properties of cells, which is of especial importance in the fields of cytochemistry, molecular biology, genetics, immunology, and biomedicine and so forth. Two major fields are environmental monitoring and point-of-care (POC) disease diagnosis including a range of applications, from drinking water quality and food pollution to epidemics and bioterrorism.
Several techniques have been used for cell analysis. One method for analyzing biological cells at high speed is flow cytometry (FCM) (2), wherein the cell samples are marked with fluorescence labels so that they can be recognized in a detection area. The samples are prepared as a cell suspension in a buffer fluid and chemically treated with one or more labels, then they are pumped through a flow channel while being kept in the center of the channel under hydrodynamic focusing control. In the detection area, the cells are illuminated by a focused laser beam and emit fluorescent light. Several detectors are placed at different angles to collect the absorption, scattering or fluorescent light. After finishing the analysis, the cells are separated into different collectors.
The traditional method for determining the refractive index of the cell is interferometry based on flow cytometry (FCM) technology (3,4). The cells to be detected are transported into the sensor zone by the hydrodynamic control and irradiated by a laser light. The laser light is reflected from different regions in the channel, which generate an interference pattern that moves when the refractive index of the flow in the channel changes. Since the cell generally has a refractive index which is different from that of the surrounding medium, the refractive index of the cell is able to be determined by the intensity variations in the interference pattern.
Unfortunately, all these techniques have their individual shortcomings. First, the cell sample requires time-consuming chemical treatment and fluorescence labeling; the fluorescence labels will change the characteristics of the natural cells; moreover, those cells labeled with fluorescent markers cannot be further used, such as for transplantation. Second, the errors of detecting scattered light from an individual cell are quite large; that is why the result from FCM is a statistic distribution and the tested sample requires certain enrichment (concentration of cells), which implies that FCM is not suitable for determining the properties of a single cell. Third, due to the low efficiency of scattering and fluorescence from the small size of biological cells (typically less than 20 μm in diameter) and also a limited area where the fluorescence labels are excited, the signal (intensity) for each cell passing through the detection area may be weak compared to the intensity of incident laser light. Therefore, the limits of sensitivity of FCM depend critically on the power of the incident laser beam and magnitude of the perturbations in the scattered or fluorescent light caused by different variants of the biological cells. Finally, a flow cytometer is a big and expensive machine; it costs normally from $200,000 to $500,000 and needs a trained operator.
Recently, Gourley (U.S. Pat. No. 5,608,519) used a vertical cavity surface emitting laser (VCSEL) as a sensor to analyze red blood cells. By placing the cells within the resonant cavity as one part of the gain medium, some parameters of the cells can be obtained by analyzing the laser beam emitted from the VCSEL. For example, the emission spectrum of VCSEL includes information on the cell ingredient because of the specific energy level structure. The cell size can be analyzed by the distribution of transversal modes. However, this method has its shortcomings. First, the fabrication for this peculiar VCSEL is very complicated and vulnerable. Making a channel within the laser die increases the difficulty of the process. Second, the dimension of the channel is limited by the size of VCSEL. Moreover, this apparatus can only analyze cells with a narrow size range under a certain channel width. Thus, big cells may be distorted in the channel and small cells might not be efficient enough in influencing the laser emission. Third, the cells to be analyzed can not be exactly located in the middle of channel without microflow control. Finally, it is very difficult to make the VCSEL resonant. Since the biological cell serves as a part of the gain medium, whether the laser will produce an emission after passing through the cells many times will be determined by the energy level structures of the cells. If the cells have a very strong absorption, the laser will not lase because of the high loss, and the cavity can not operate in resonance.
Accordingly, while prior art devices allow for analyzing cells, it is desirable to improve these devices by having systems which are more accurate, work with a single operation, have low costs, and high accuracy.